Differentiation-induced reduction in functional diversity restricts the ability of cytomegalovirus-specific CD8 T cells to eliminate virus-infected cells

Individuals with expanded CD8 T cells recognizing the pp65-HLA-A*02:01–restricted viral epitope NLVPMVATV (NLV-T cells) have weakened immune control of human cytomegalovirus (HCMV) reactivation. Here, we characterized NLV-T cells from healthy HCMV-positive donors, dividing them into two groups: those with low and those with high NLV-T cell frequencies (LF and HF, respectively). Our comprehensive multimodal analysis revealed that NLV-T cells from HF donors preferentially exhibit a phenotype of advanced differentiation, characterized by granzyme B and perforin expression. Although these cells were more efficient in eliminating peptide-loaded target cells than NLV-T cells from LF donors, the latter were more potent in cytokine secretion and the elimination of HCMV-infected cells with virus-induced HLA class-I downregulation. Overall, these findings suggest that HCMV exploits CD8 T cell differentiation to evade immune protection and explain the previously observed decline in HCMV reactivation control in individuals with NLV-T cell accumulation. Overall design: For single cell RNA seq: PBMCs were thawed and either used for staining directly (HF donors) or CD8+ T cells were enriched using magnetic activated cell sorting (MACS) (LF donor samples). The PBMCs from HF donors and the enriched CD8+ T cells from LF donors were stained with NLV-peptide loaded MHC class I tetramers for 15 minutes at 37°C, followed by staining for extracellular markers, Totalseq antibodies, and cell-hashing antibodies (Table 4) for 25 minutes at RT. The samples were washed and up to 8 samples were pooled and cells were sorted for CD19-, CD14-, CD16-, CD56-, CD3+, CD8+, NLV-tetramer+ cells on a Becton-Dickinson FACSAria III Fusion or Becton-Dickinson FACSAria IIu cell sorter. For bulk RNA and TCR sequencing: The NLV-T cells were expanded via NLV-peptide stimulation for 9 days and frozen. For sequencing, the cells were thawed and the NLV-T cells were isolated with NLV-loaded MHC class I streptamers.